Results
Due to complications with amplification and assembly of synthetic sequences of genes that we ordered like complex stable secondary structures and a lot of long repeats we switched to using plasmids that we received from as a kind gesture from Daniel C. Ducat. Plasmid p233 that contains hydrogenase maturation factors HydEF and HydG under psbA1 promoter and HydA under psbA1 promoter in 0015 plasmids.
To assemble psbA1 and HydA genes, firstly, amplification of psbA1 (503 bp) from p233(11830 bp) and HydA(1998 bp) from plasmid PAM (5004 bp) were done. As it can be seen from Figure 1 and Figure 2 successful amplification of genes of interest was verified via gel electrophoresis.
Figure1: PsbA1 amplification gel results.
Figure2: HydA amplification gel results
Amplified HydA and psbA1 genes were assembled via Polymerase Cycling Assembly (PCA) and also verified via gel electrophoresis as shown on Figure 3.
Figure 3. HydA and PsbA1 assembly gel results
After transformation of the assembled construct into the DH5alpha cloning strain, DNA extraction results illustrated bands higher than empty vector p233 (also known as MF (Maturation Factor)). All samples were linearized with BamHI-HF beforehand.
Figure4. Linear assembled construct digested by BamH gel results
To ensure the presence of HydA gene in the construct, the samples were linearized by EcoRV and used as template for HydA amplification (see Figure 5). Overall, four samples were assessed for the HydA and three were positive for the gene as bands corresponding to ~1.7-1.8 kb were visualized on the gel. As a negative control p233 (also known as MF) without insert was used which did not show any band for HydA as expected.
Figure5. HydA verification in the construct
To verify the integration of construct into cyanobacteria psbA1+hydA(~3.1 kb) amplification was performed using forward psbA1 and reverse NSb primers. NSb primer was used due to complications during amplification process with HydA primers. Out of five samples, the 5th band without ladder had an expected band for ~3.1 kb.
Figure6. HydA+PsbA1 amplification from the construct integrated into cyanobacteria
The effect of genetic modification on survivability of cyanobacteria in a hydrogen sulfide (H2S) rich and anaerobic environment was assessed qualitatively. The samples of nearly same OD were constantly shaken in the shaking incubator. From Figure 7, it can be seen that transformed HydA+ cells differ significantly from the wild-type by its color illustrating the higher survival rate compared to the wild-type strains.
Blotted paper was also assessed using survival assay with the same condition only few adjustments for the paper. It was soaked in the medium with sulfide and w/o sulfide. Tubes were flushed with nitrogen until air is replaced. 5 days later tubes were assessed on paper colour to confirm survival.
As Ducat stated limited growth is supported in Cyanobacteria as they use Hydrogen as reductant in alternative metabolic pathways. This is supported by this survival assay and thus hydrogen presence by HydA activity.
Figure 7. Sulfide survival assay
Pop test was performed as a classical way to detect hydrogen in the air one of our tubes showed exact same result to a lesser extent as the control reaction of Zinc + Hydrochloric Acid. Zinc + HCl reaction was done in the same sealed anaerobic tube. Stopper was punctured using need and excess gas was let out straight to the flame of match instead of usual “Pop” sound we heard whistle in both cases and same reaction of flame to the gas that went out in control and our culture tube. This leads us to be sure that hydrogen is being produced:
Control reaction video:
Transformed cells video:
Wild type video:
Hydrogen gas production measurement
There were two cultures cultured in two different times (04.10.19 and 16.10.19). They were flushed by nitrogen gas in order to make anaerobic conditions. The pressure and temperature of samples were measured on 22.10.19. The Ideal Gas Apparatus, PASCO sensor PASCO Capstone software were used to measure pressure and temperature of the system. Below, you can see the photo of our measurement setup:
Figure 8. Gas measurement apparatus
We collected gas sample from our cultures grown in anaerobic conditions into the special measuring cylinder. We measured the volume of the collected gas, then we applied pressure by pushing down the plunge. This pressure was measured by special Pasco detectors. From the Pasco Capstone software, we obtained the values of pressure and temperature. Using the Ideal Gas equation, we measured the number of moles:
PV = nRT
Volume contains the parasitic volume of apparatus.
In order to compensate for the difference of OD, we obtained a ratio of number of moles of gas produced per OD of culture measured.
For the Wild type cultures we obtained the following graph:
Figure 9. The hydrogen production measurements for the Wild Type
For the Transformed cultures:
Figure 10. The hydrogen production of the transformed cyanobacteria (SBA21)
As it can be seen, there is an increase in gas production by transformed cyanobacterial cultures, and clear decrease of gas produced by the wild type.
In our measurement, we constructed the experiment by variating the parameter of H2S presence. H2S is known to be the common PSII inhibitor, which we are aiming to accomplish. This fact should increase the production of Hydrogen gas. We did the measurement for two types of cultures, SBA21 and HydA1, that were transformed in different days. We also performed the same measurement with Wild type.
Figure 11. Hydrogen production by various cell cultures
Error Propagation
As it can be seen, Wild type does not deal with the survival in the presence of H2S, and that is why both proliferation and hydrogen gas are greatly inhibited. On the other hand, there is a clear difference in the production of gas for the transformed cultures. H2S increases yield for the gas production for both HydA1 and SBA21, and works as we expected. Beside of this, there is clear increased hydrogen production comparing to the Wild type in overall. The increased hydrogen production in the presence of H2S proves that our part works as expected.
Survival test of bacteria on filter paper
Bacteria were fixed on the filter paper by mixing equal amount of latex and culture, and placing dropwise on the paper, and solution of BG11 was poured until it topped the paper. Initially, the pH of latex was measured by a pH-meter, but the value was quite fluctuating. Therefore, it was decided to use pH-indicator strips. The resultant pH was 6. This was quite acceptable for the culture. Starting from the first day, the BG11 solution was being changed everyday. This was done due to the high mass convection inside hood. However, this caused high consumption of BG11. Therefore, it was decided to put larger plate on top of the plate with filter paper, to prevent evaporation of BG11. On average, in 3 weeks, the color of bacteria changed from light green to pale yellow, meaning the life expectancy was about 2 weeks considering equal amounts of culture and latex, exposure to surroundings, and slow evaporation rate of BG11.
Figure 12. The survival test of bacteria on the filter paper.
Carbon Quantum Dots
Microalgae C. reinhardtii containing chromate reductase gene
Six cultures named 62, 53, 58A, 58B, C and wild type were checked for the presence of chromate reductase (ChrR) gene by using PCR on liquid cultures.
Figure 13. ChrR gene extraction from cultures.
From this, we could identify the presence of the gene in several strains and chose the corresponding biomass for preparing Carbon Quantum Dots.
The CQDs were prepared from the strains containing Chromate reductase gene. We performed excitation wavelength-dependent fluorescence measurements to detect the optimum wavelength. We excited the solutions of CQDs with the range of 350-450 nm and obtained the results presented on figure 14.
Figure 14. 3D data for the fluorescence of CQDs
As it can be observed, the fluorescence properties of our CQDs can be tuned upon different excitation wavelengths. Based on this study, we identified that most pleasurable fluorescence is obtained upon the excitement with 425 nm. The graph is presented on figure 15.
Figure 15. Fluorescence of CQDs excited with 425 nm
The maximum peaks are observed on 482, 611, 661 nm. The first peak overlaps with the absorption of Gloebacter Rhodopsin which is in 500 nm range, and the third one is close to the absorbance of the PSI which is in the range of 700 nm.
We also coated cyanobacteria strains with Carbon Quantum Dots and performed the analysis with the Confocal Microscopy, using the 409 nm excitation wavelength.
Figure 16. The picture of coated cyanobacteria from the confocal microscopy
As it can be seen, half of the cyanos are coated with fluorescent CQDs.
We also performed time-resolved fluorescence measurement, and using the Decay Fit software we modeled the major fluorescent events occurring with CQDs. The modeling description can be seen in detail on the Modeling web page.
